Poor sanitation infrastructure in rural areas can often lead to high levels of fecal contamination in local waterbodies and subsequent exposure to waterborne disease can occur. Although standard water quality measures such as quantification of E. coli can reveal relative concentrations of fecal contamination, they do not pinpoint the sources of such contamination. Source assessment in rural areas affected by untreated household waste might be improved with the human-specific, microbial source tracking marker HF183. This study attempted to quantify HF183 in two particular Appalachia streams with known discharges of untreated household waste. Water samples were taken above and at multiple points below these discharges on 29 occasions between August 2012 and April 2016, and tested for both HF183 and E. coli. HF183 was detected consistently in one of the study streams, though the concentrations were generally much lower than those previously reported in raw sewage; in the other watershed, HF183 was never detected. Further analysis via a multiple linear regression model showed a positive correlation between the level of E. coli and the proximity and number of known waste discharge points upstream from each sampling site. Primary conclusions of this study include: 1) HF183 is not always detected, even in watersheds with known sources of human fecal contamination, 2) it may be a useful water quality assessment tool where such contamination is suspected, particularly in cases where contaminant source allocation is necessary for setting mitigation priorities. / Master of Science

Many Neotropical felid species, such as jaguars, are threatened with extinction due to habitat fragmentation and/or human persecution. Human activities around protected areas in Belize, Central America, are increasing and so are levels of human-felid conflict. Potential consequences of this conflict are an increase in stress impacting health, diet shifts, or heightening of animal aggression. The goal of this work was to assess the effects of human-modified habitats on native felids by comparing fecal glucocorticoid metabolite (FGM) concentrations, endoparasite species richness (ESR), and diet using non-invasive scat sampling in a protected forest vs. surrounding non-protected areas in Belize. Field studies relying on non-invasive fecal hormone monitoring are subject to potential hormone degradation in samples exposed to the environment. Therefore I conducted immunoassay and environmental validations for measuring FGM in jaguars (Panthera onca). In the field, I collected scat using a detector dog, identified samples using DNA, retrieved parasite propagules with a flotation technique, and identified prey remains by morphology. I detected five felids: jaguar, puma, ocelot, jaguarundi and domestic cat. FGM concentrations were higher in pumas and jaguarundis than in the other felids. I found no livestock remains in felid scats. ESR was similar across felid species. Domestic cats were found only in human-modified areas. This results provide a baseline on adrenal activity, prey consumption, and endoparasites in felids of Belize. These findings could be used for comparisons to populations thought to be affected by human activities across Belize and in neighboring countries. / Master of Science

Intestinal flora of wild chimpanzee has not been studied. Fecal flora analyses currently give insight to this environment. We collected feces from twelve human-habituated wild chimpanzees in each of three age groups: four juveniles, four sub-adults, and four adults. We analyzed fecal samples using Terminal-Restriction Fragment Length Polymorphism (T-RFLP) of amplified 16S rRNA genes to determine bacterial diversity present. Between 1 and 14 terminal-restriction fragments (T-RFs) were observed in each sample. A total of 26 unique T-RFs were produced from the samples and ranged in size from 92 to 837 base pairs (bps). Twenty-four of these T-RFs corresponded to five bacterial phyla: Actinobacteria, Bacteroidetes, Firmicutes, Mollicutes, and Proteobacteria, as well as uncultured and unidentified bacterial species. The remaining T-RFs corresponded solely to uncultured or unidentified bacteria. Firmicutes was the most common phylum, observed in 11 of the samples. Bacteroidetes was the second-most common phylum, detected in 8 of the samples. Principal Components Analysis (PCA) revealed a discrete clustering of 10 samples when looking at components one and two, and a clustering of 11 samples when looking at component three. These three components accounted for 72.5% of the variation within the data. Morisita indices were computed to compare T-RF profiles of two samples at a time, and were between 0 and 0.886. Results indicated that some fecal bacterial profiles were similar in the study group, but ultimately varied between samples when compared two at a time. Specific diet, physiology, and environmental reservoir exposure may play large roles in shaping such profiles. / Master of Science

Transport of fecal bacteria and nutrients from point and nonpoint sources to surface water bodies is of significant concern in Virginia and the United States. In Virginia, 4,320 river miles are impaired for one or more beneficial use and 72% of the streams are impaired due to pathogen indicators (VDEQ, 2002). Land applications of manure from confined animal systems and by direct deposit by grazing animals are both major sources of fecal bacteria and nutrients in runoff. Therefore, an understanding of the overland transport mechanisms for fecal bacteria and nutrients is very important for the development of best management practices to reduce loading of pathogens and nutrients to surface water bodies. The objectives of this study were to quantify the release and transport potential of three fecal bacterial indicators: E. coli, Enterococcus, and fecal coliforms; and nitrogen and phosphorus from land applied manure during runoff events. Another objective was to identify the Enterococcus species present in dairy manure and determine which species have the highest potential to be transported by runoff. Release plots were established to study the in-field bacteria and nutrient release. The bacteria and nutrients released from the plots are available to be transported to the edge of the field in runoff. Four manure treatments (turkey litter, liquid dairy manure, cowpies, and none or control) and three land type treatments: pasture with a history of poultry litter application (Turkey Farm), pasture with a history of liquid dairy manure application (Dairy Farm), and pasture with no prior manure application (Tech Research Farm) were studied. During a short but intense rainfall event, the highest bacterial release was measured under the cowpie treatment (E. coli concentrations ranging from 37,000 to >300,000 and FC concentrations ranging from 65,000 to >300,000). Pasturelands with a history of previous manure applications did not release higher bacteria concentrations compared with pasturelands which had never received manure applications. Pasturelands with a history of land application of liquid dairy manure and turkey litter had 143% and 94% higher TSS concentrations available to be transported off the field during overland flow events because of the build up of organic material on the soil surface. TP concentrations released from the cowpie, liquid dairy, and turkey litter treatments were 3.12 mg/L, 3.00 mg/L, and 1.76 mg/L, respectively. Transport plots were developed to measure the concentrations of fecal bacteria and nutrients present in overland flow at the edge of the field. The bacteria flow-weighted concentrations were highest in runoff samples from the plots treated with cowpies (200,000 CFU/100 mL of E. coli and 234,000 CFU/100 mL of FC). The turkey litter had the highest concentration of dissolved phosphorus in runoff from pasturelands (1.22 mg/L), but the cowpie treatment had the highest concentrations of sediment bound phosphorus in runoff (0.73 mg/L). All three treatments investigated in this study contributed to phosphorus loading in surface waters and could potentially increase the risk of eutrophication. Total nitrogen concentrations from the transport plots exceeded the threshold for likely eutrophication problems for all treatments and the total nitrogen concentrations from plots treated with cowpies exceeded the threshold for severe eutrophication problems. The Biolog System, a method of bacterial source tracking, was used to identify the different species of Enterococcus present both in the cowpie source manure and in the runoff collected from the transport plots treated with cowpies. The source manure is dominated by the Enterococcus mundtii (55%), Enterococcus gallinarum (20%), Enterococcus faecium (10%), and Enterococcus faecalis (10%). Enterococcus faecalis had the highest percentage of isolates present in runoff with a total of 37%, followed by Enterococcus mundtii which was present in 21% of the runoff events and Enterococcus gallinarum and Enterococcus faecium (11%). Improvements in understanding the bacterial release and overland processes will enhance modeling of bacteria and nutrient transport, and provide a basis for a more realistic evaluation of the impacts of management practices implementation. The data from this study will serve as a baseline to model the release and transport of fecal bacteria and nutrients from agricultural watersheds to surface waters. / Master of Science

During periods of reduced precipitation, flow in low-order, upland streams may be reduced and may stop completely. Under these "low flow" conditions, fecal bacteria directly deposited in the stream dominate in-stream bacteria loads. When developing a Total Maximum Daily Load (TMDL) to address a bacterial impairment in an upland, rural watershed, direct deposit (DD) fecal bacteria sources (livestock and wildlife defecating directly in the stream) often drive the source-load reductions required to meet water quality criteria. Due to limitations in the application of existing watershed-scale water quality models, under low-flow conditions the models can predict unrealistically high in-stream fecal bacteria concentrations. These unrealistically high simulated concentrations result in TMDL bacteria source reductions that are much more severe than what actually may be needed to meet applicable water quality criteria. This study used the Hydrological Simulation Program-FORTRAN (HSPF) to compare three low-flow DD simulation approaches and combinations (treatments) on two Virginia watersheds where bacterial impairment TMDLs had been previously developed and where low-flow conditions had been encountered. The three methods; Flow Stagnation (FS), DD Stage Cut-off (SC), and Stream Reach Surface Area (SA), have all been used previously to develop TMDLs. A modified version of the Climate Generation (CLIGEN) program was used to stochastically generate climate inputs for multiple model simulations. Violations of Virginia's interim fecal coliform criteria and the maximum simulated in-stream fecal coliform concentration were used to compare each treatment using ANOVA and Kruskal Wallis rank sum procedures. Livestock DD bacteria sources were incrementally reduced (100%, 50%, 15%, 10%, 5%) to represent TMDL load reduction allocation scenarios (allocation levels). Results from the first watershed indicate that the FS method simulated significantly lower instantaneous criterion violation rates at all allocation levels than the Control. The SC method reduced the livestock DD load compared to the Control, but produced significantly lower instantaneous criterion violation rates only at the 100% allocation level. The SA method did not produce significantly different instantaneous criterion violation rates compared to the Control. Geometric mean criterion violation rates were not significantly different from the Control at any allocation level. The distributions of maximum in-stream fecal coliform concentrations simulated by the combinations SC + FS and SC + SA + FS were both significantly different from the Control at the 100% allocation level. The second watershed did not produce low-flow conditions sufficient to engage the FS or SC methods. However, the SA method produced significantly different instantaneous violation rates than the Control at all allocation levels, which suggests that the SA method continues to affect livestock DD loads when low-flow conditions are not simulated in the watershed. No significant differences were found in the geometric mean violation rate or distribution of maximum simulated in-stream fecal coliform concentrations compared to the Control at any allocation level. This research suggests that a combination of the SC and FS methods may be the most appropriate treatment for addressing unrealistically high concentrations simulated during low-flow conditions. However, this combination must be used with caution as the FS method may increase the maximum simulated in-stream fecal coliform concentration if HSPF simulates zero volume within the reach. / Master of Science

Determining intake of feeds in horses is an important factor in incorporating supplements in their diets. Fecal recoveries (R), fecal output (FO), dry matter digestibility (DMD) and dry matter intake (DMI) were estimated using alkanes as markers in 8 thoroughbred geldings. The experiment compared two diets in a 2 X 2 latin square experiment. The diets were mixed grass hay only (H) and the same hay plus a cereal by-product (H + CBP). The cereal by-product (CBP) was the high fat component added to feeds at Virginia Tech's Middleburg Agricultural Research and Extension Center. The apparent digestibility of ether extract (EE) and other nutrients in the H and H + CBP, as well as the partial digestibility of CBP were also determined. The periods were 21 d each with a dietary accommodation period followed by eight days of dosing the even chain alkanes dotriacontaine (C32) and hexatriacontane (C36) as external markers. Total collection (TC) was performed the last 4 d of dosing. The results show that mean recoveries of alkanes were close to 100%, but the range for individual alkanes was wide, and the pattern of recoveries for alkanes of different chain length was inconsistent from feed to feed. The results also indicate that mean estimates of the DMI, DMD and FO of a feed, such as H or H + CBP, are determined with reasonable accuracy by means of alkane markers. In contrast, alkane estimates of DMI and DMD in an individual horse fail to predict corresponding TC estimates. The alkane estimate of FO in an individual horse predicts a TC value with error of 16.4%. The CBP was found to be an excellent source of EE, CP and fiber but a poor source of Ca. / Master of Science

Fecal bacteria and pathogens are a major source of surface water impairment. In Virginia alone, approximately 73% of impaired waters are impaired due to fecal coliforms (FC). Because bacteria are a significant cause of water body impairment and existing bacterial models are predominantly based upon laboratory-derived information, bacterial models are needed that describe bacterial die-off and transport processes under field conditions. Before these bacterial models can be developed, more field-derived information is needed regarding bacterial survival and transport. The objectives of this research were to evaluate bacterial survival under field conditions and to develop a comprehensive, spatially variable (distributed) bacterial model that requires little or no calibration. Three field studies were conducted to determine die-off or diminution (settling plus die-off) rates of FC and Escherichia coli (EC) over time in: 1) dairy manure storage ponds and turkey litter storage sheds, 2) pasture and cropland soils to which dairy manure was applied, and 3) beef and dairy fecal deposits. The dairy manure storage ponds were sampled just under the pond surface. The FC and EC diminution (settling plus die-off) rates for dairy manure storage ponds were 0.00478 day⁻¹ and 0.00781 day⁻¹, respectively. The five samples collected for turkey litter in storage were inadequate to draw any conclusions. Bacterial die-off rates in cropland and pastureland soils were found to be statistically different from each other at the α = 0.05 level. The FC and EC die-off rates in cropland soils were 0.01351 day⁻¹ and 0.01734 day⁻¹, respectively, while the FC and EC die-off rates in pastureland soils were 0.02246 day⁻¹ and 0.02796 day⁻¹, respectively. Die-off rates for bacteria from dairy heifer, dairy milker, and beef cow fecal deposits were not statistically different from each other. The resulting die-off rate constants for fecal deposits were 0.01365 day⁻¹ and 0.01985 day⁻¹ for FC and EC, respectively. The EC/FC ratio was also evaluated for the fecal deposits and land-applied manure to determine if a quantifiable relationship was discernable. In general the EC/FC ratio declined over time, but no quantifiable relationship was discerned. The bacterial model simulates die-off, bacterial partitioning between soil and water, and bacterial transport to surface waters in free (in solution) and sediment-adsorbed forms. Bacterial die-off was modeled using Chick's Law, bacterial partitioning was modeled with a linear isotherm equation, and bacterial transport was modeled using continuity and flow equations. The bacterial model was incorporated into the ANSWERS-2000 model, a continuous, distributed, nonpoint source pollution model. The model was tested using data from two plot studies. Calibration was required to improve runoff and sediment predictions. Bacterial model predictions underpredicted bacterial concentrations in runoff with a maximum underprediction error of 92.9%, but predictions were within an order of magnitude in all cases. Further model evaluation, on a larger watershed with predominantly overland flow, over a longer time period, is recommended, but such data were not available at the time of this assessment. The overall conclusions of this research were 1) FC and EC die-off or diminution under the examined field conditions followed Chick's Law, 2) measured die-off rate constants in the field were much less than those cited in literature for laboratory experiments, and 3) for the conditions simulated for two plot studies, the bacterial model predicted bacterial concentrations in runoff within an order of magnitude. / Master of Science

Antibiotic resistance analysis (ARA) was used to determine sources of fecal pollution in the Blackwater River in South-central Virginia. The Department of Environmental Quality designated six segments as impaired due to high fecal coliform concentrations with non-point source (NPS) agriculture the suspected source of impairment. The Blackwater River watershed encompasses 72,000 ha of dairy, beef, and intensive production agriculture, abundant wildlife populations and many homes with onsite septic systems. A library of antibiotic resistance profiles based on 30 concentrations of 9 antibiotics was developed for 1,451 enterococci isolates from human, cattle, chicken, horse, goat, sheep, deer, raccoon, muskrat, goose, duck, coyote, and wild turkey fecal samples. Each isolate was classified as human, wildlife or livestock. Correct classification rates were 82.3% for human, 86.2% for livestock and 87.4% for wildlife isolates when profiles were analyzed with discriminant analysis. Profiles were also determined for 48 isolates from 128 stream samples collected periodically from August 1999 thru April 2001 and compared to the known sources using discriminate analysis. A human signature was found at each site at least once during the year, ranging from 0.0% to 85.0% of the sample isolates. The livestock signature varied from 2.3% to 100% over sites and months, and the wildlife signature varied from 0.0% to 79.5%. The results indicate that both humans and wildlife contribute to fecal pollution in addition to the suspected source, livestock, and reducing fecal pollution will require consideration of all three sources. The results from this research are being used to develop a total maximum daily load (TMDL) project allocations for fecal coliforms in the Blackwater River. Isolates identified by ARA were also profiled using the Biolog metabolic identification system. A library of metabolic profiles was constructed from known source isolates. Stream isolates were identified by Biolog and the metabolic profile was compared to the Biolog library. Of ten stream isolates identified by ARA as human, the Biolog library identified one as human, four as livestock, and five as wildlife. Of ten isolates identified by ARA as livestock, the Biolog library identified seven as livestock and three as wildlife. Of ten isolates identified by ARA as wildlife, one was identified as human, three as livestock and six as wildlife. The overall correct classification of Blackwater isolates in the Biolog library was 14 of 30 isolates, or 47%. Although the Biolog library was constructed with some isolates from the Blackwater basin, there may not be enough isolates in the Biolog library to adequately represent the variability shown by the Blackwater isolates, resulting in lower than expected correct classifications. In spite of these results, Biolog remains promising as one of several tools with potential as a bacterial source tracking method. / Master of Science

[pt] O presente trabalho tem como objetivo investigar a contaminação por esgotos domésticos do sistema estuarino formado pelo rio Iguaçu e canal de Sarapuí, localizados na porção noroeste da Baía de Guanabara, através da determinação de esteróis e composição elementar (C e N) da matéria orgânica em amostras de material particulado em suspensão e sedimentos, e da caracterização físico-química e química da água e sedimento. As amostragens foram realizadas em março/2004 e setembro/2004, em 7 estações de água e 10 de sedimento, distribuídas entre 6 km a montante do rio Iguaçu e 4 km dentro da baía. Também foi coletada uma amostra composta de esgoto bruto na Estação de Tratamento da Ilha do Governador (ETIG). Na determinação dos parâmetros foram utilizados métodos analíticos já estabelecidos, tais como cromatografia a gás/espectrometria de massa (esteróis) e oxidação a seco (C e N), após otimização para as condições específicas de campo e de laboratório. As concentrações dos principais esteróis variam em escala temporal e espacial, em função de fatores climáticos e da localização de fontes difusas de esgoto. Há gradientes decrescentes de concentração de esteróis entre rio e a baía, com predomínio de esteróis fecais sobre fitoesteróis. Coprostanona (5b-colestan-3b-ona) atingindo concentrações de até 21 mg gC-1, epicolestanol (5a-colestan-5a-ol) e coprostanol (5b-colestan-3b- ol) foram os mais abundantes. A detecção de epicolestanol (inédita para a baía de Guanabara) em concentrações elevadas no material particulado e sedimento (assim como no esgoto bruto), as diferenças na composição entre esteróis no esgoto bruto em comparação com o material particulado e sedimento, e os resultados das razões entre determinados esteróis, são resultados que refletem a importância dos processos bacterianos em alterar a composição original da matéria orgânica e dos esteróis associados. O conjunto de informações levantadas confirma o elevado nível de degradação ambiental da região estudada. Por outro lado, a dinâmica do sistema e processos pré- e pós- deposicionais influenciam na geoquímica dos esteróis e, portanto, seus efeitos devem ser levados em consideração em estudos sobre marcadores moleculares em estuários com características semelhantes ao do presente trabalho. / [en] The contamination by domestic sewage of the Iguaçu River and Sarapuí channel, located in the north-western sector of Guanabara Bay, was investigated by the determination of sterols and elemental composition (C and N) of organic matter in suspended matter and superficial sediments. Physico-chemical and chemical characterization of water and sediments were considered as well. Conventional analytical methods, like gas chromatography/mass spectrometry (sterols) and dry chemical oxidation (C and N), were employed after optimization for specific field and laboratory conditions. In March and September/2004 water/suspended particles (7 sample stations) and sediment (10 stations) were collected in a transect from 6 km up the river until 4 km off the river, in the bay. An additional sample of bulk sewage was also collected at a sewage treatment plant close to the studied area. The faecal sterols predominate over phyto sterols in suspended matter and sediments. Among the 14 sterols quantified, higher concentrations (up to 21 mg gC-1) were measured for coprostanone (5b-cholestan- 3b-one), epicholestanol (5a-cholestan-3a-ol) and coprostanol (5b-cholestan-3b- ol). In general, there is a river-bay decreasing gradient in sterol concentration, associated with the location of diffuse sources of sewage. Seasonal variation in climatic factor also influenced in the sterols distribution. The presence of epicholestanol (reported here for the first time in Guanabara Bay) in relatively high concentrations in suspended matter and sediments (as well as in raw sewage), the differences in sterol composition between raw sewage and suspended matter/sediments, and the values obtained for selected sterols source-diagnostic ratios, suggested the occurrence of significant microbial alteration of organic matter, with implications on the geochemistry of sterols. The elevated degradation

As leveduras Saccharomyces cerevisiae são consideradas importantes matérias primas na nutrição animal pela sua capacidade prebiótica. Os prebióticos são compostos não digeridos pelo organismo animal, mas que são fermentados pelos microrganismos do trato gastrintestinal, cujos produtos são capazes de prover benefícios ao hospedeiro. A fermentação depende de fatores como: substrato utilizado para o seu crescimento, método de fermentação, modo e condição de secagem e idade das células. Assim, os processos produtivos modernos têm como intuito a produção de leveduras com elevado potencial prebiótico. Este estudo objetivou avaliar os efeitos de teores crescentes de leveduras com metabólitos ativos (LSC; baseada na fermentação de substratos específicos) na digestibilidade aparente dos nutrientes da dieta, microbiota e produtos da fermentação fecal e parâmetros imunológicos de gatos adultos. Foram utilizados 27 gatos adultos, idade média de 9,44±5,35 anos, machos e fêmeas, sem raça definida e saudáveis. Os animais foram distribuídos em delineamento em blocos casualizados (idade), constituído de três tratamentos experimentais, denominados: DC (dieta controle), LSC 0,3 (dieta controle com 0,3% de leveduras com metabólitos ativos) e LSC 0,6 (dieta controle com 0,6% de leveduras com metabólitos ativos). Os resultados obtidos foram analisados através do programa computacional Statistical Analysis System (SAS Institute Inc., 2004), sendo considerados significativos valores de p<0,05 e as médias comparadas pelo teste de Tukey. Verificou-se que a inclusão do aditivo alterou apenas a digestibilidade aparente da fibra bruta, da matéria mineral e energia metabolizável (p<0,05). Já em relação aos produtos da fermentação e microbiota das fezes, observou-se redução em ácido lático (p=0,0040) e Clostridium perfringens (p=0,0226) com a inclusão do prebiótico e diminuição do ácido isovalérico (p=0,0144) no tratamento LSC 0,3. Pode-se concluir que o aditivo, nos teores de inclusão avaliados, parece apresentar potencial prebiótico em relação aos produtos da fermentação e microbiota fecal / Saccharomyces cerevisiae yeast are considered important raw materials in animal nutrition due their prebiotic capacity. Prebiotics are compounds not digested by animal organism, but are fermented by microorganisms in the gastrointestinal tract, which products are capable of providing benefits to the host. The fermentation depends on factors such as: substrate used for growth, fermentation method, way and drying condition and age of the cells. Thus, modern production processes have the objective of producing yeast with high prebiotic potential. This study aimed to evaluate the effects of increasing levels of yeast with active metabolites (LSC) based on the fermentation of specific substrates on apparent digestibility of diet nutrients, microbiota and fecal fermentation products and immunological parameters in adult cats. Twenty seven male or female cats with mean weight of 4.19 ± 0.83kg and mean age of 9.44 ± 5.35 years were used and distributed in an unbalanced randomized block design (age), consisting of three experimental treatments, DC (control diet), LSC 0.3 (control diet with 0.3% yeast with active metabolites) and LSC 0.6 (control diet with 0.6% yeast with active metabolites). The results were analyzed using the computer program Statistical Analysis System (SAS Institute Inc., 2004), with significance level of p<0.05 and the averages compared by Tukey test. The inclusion of the additive only changed the apparent digestibility of crude fiber and mineral content (p<0.05). Regarding the fermentation products and microbiota of feces, there was a reduction in lactic acid (p=0,0040) and Clostridium perfringens (p=0,0226) with inclusion of prebiotic and decreased of isovalerate (p=0,0144) in the LSC 0.3 treatment. It can be concluded that the additive, in the levels of inclusion assessed, seems to have prebiotic potential on fecal fermentation products and microbiota